1. Field of the Invention
This invention is concerned with diagnostic methods which assist in classification of tumors by phenotype and with therapeutic intervention tailored to particular tumor phenotypes. In particular this invention concerns mutant forms of type II receptors (RII) for transforming growth factor beta (TGFxcex2) which inactivate growth suppression by TGFxcex2, methods for detecting inactivation of TGFxcex2 RII receptor and therapeutic methods for restoring tumor suppression by restoring RII faction.
2. Review of Related Art
TGFxcex2 inhibits growth of multiple epithelial cell types, and loss of this negative regulation is thought to contribute to tumor development (Roberts, et al., 1990, In Peptide Growth Factors and Their Receptors. Handbook of Experimental Pharmacology, A. Roberts and M. Sporn, Eds., (Springer-Verlag, Heidelberg) pp. 419-472; Massaguxc3xa9, J., 1990, Annu. Rev. Cell Biol., 6:597; Moses, et al., 1990, Cell, 63:247; Filmus, et al., 1993, Curr. Opin. Oncol., 5:123; Markowitz, S., et al., 1994, J. Clin. Invest., 93:1005; Wu, S., et al., 1992, J. Cell. Biol., 116:187; Wu, S., et al., 1993, Cell Growth Differ., 4:115; Park, J., et al., 1994, Proc. Natl. Acad. Sci. U.S.A., 91:8772; Manning, et al., 1991, Oncogene, 6:1471; Hoosein, N., et al., 1989, Exp. Cell Res., 181:442; Geiser, et al., 1992, J. Biol. Chem., 267:2588). Previous studies have demonstrated that TGFxcex2 suppresses growth of certain cancer cell lines, that antisense inhibition of TGFxcex2 enhances the tumorigenicity of weakly tumorigenic cancer cell lines, and that certain tumor cells can become unresponsive to TGFxcex2 (Markowitz, S., et al., 1994, J. Clin. Invest., 93:1005; Wu, S., et al., 1992, J. Cell. Biol., 116:187; Wu, S., et al., 1993, Cell Growth Differ., 4:115; Park, J., et al., 1994, Proc. Natl. Acad. Sci. U.S.A., 91:8772; Manning, et al., 1991, Oncogene, 6:1471; Hoosein, N., et al., 1989, Exp. Cell Res., 181:442; Geiser, et al., 1992, J. Biol. Chem., 267:2588).
The TGFxcex2 growth inhibitory signal is transduced through two receptors, type I (RI) and type II (RII) which function as a heteromeric complex (Lin, et al., 1992, Cell, 68:775; Moustakas, A., et al., 1993, J. Biol. Chem., 268:22215; Wrana, J., et al., 1992, Cell, 71:1003).
It is an object of this invention to provide a method for diagnosis or prognosis of cancer by detection of the absence of functional TGFxcex2 receptor RII in cells of a patient.
It is another object of this invention to provide a nucleotide sequence encoding a mutant form of RII, to provide mutant RII protein and to provide antibodies specifically immunoreactive with mutant RII.
It is yet another object of this invention to provide therapeutic methods for treating tumors having inactive RII by replacement gene therapy.
It is still another object of this invention to provide immunogenic compositions which elicit antibodies specifically reactive with cells expressing mutant forms of RII.
These and other objects are provided by one or more of the following embodiments.
In one embodiment, this invention provides a diagnostic method to aid in predicting prognosis of a colon or gastric cancer patient comprising determining the quantity of functional type II receptor for TGFxcex2 (RII) in cells from tumor tissue of the patient, and comparing the quantity of RII in tumor cells to the quantity of RII in non-neoplastic cells of the patient, reduced RII in the tumor cells being indicative of altered prognosis. In a particular embodiment, this invention provides a screening method to aid in classifying tumor cell phenotype in a patient comprising determining the presence or absence of functional RII receptor for TGFxcex2 in tumor tissue from the patient, the absence of functional RII being indicative of carcinoma with replication errors (RER phenotype). The screening method is preferably applied to tumor tissue from endometrial cancer, ovarian cancer, gastric cancer, or pancreatic cancer, most preferably to colon cancer.
In another embodiment, this invention provides a method to aid in diagnosing cancer in a patient comprising detecting, in the patient, a non-functional mutant form of a growth regulatory gene, wherein the growth regulatory gene encodes a type receptor which is a member of a family of serine/threonine receptors which bind members of a family of TGFxcex2-like factors or the wild-type of the growth regulatory gene contains repetitive DNA sequence motifs in the coding region, presence of the non-functional mutant form of the growth-regulatory gene being indicative of tumor tissue or precancerous lesions in the patient. In a particular embodiment, this invention provides a screening method to aid in diagnosing cancer in a patient comprising determining the presence or absence of functional RII receptor for TGFxcex2 in tissue from the patient, the absence of functional RII being indicative of tumor tissue or precancerous lesions in the patient. In a particular embodiment, the screening method comprises determining the presence or absence of mutant forms of RII receptor for TGFxcex2, preferably mutant forms of RII encoded by RII cDNA altered by a mutation selected from the group consisting of a two base pair insertion of GT which occurs in a six base pair repeat sequence GTGTGT at nucleotides 1931-1936 of the normal RII sequence, deletion of one A base pair from a ten base pair poly A sequence at nucleotides 709 to 718 of the normal RII sequence, deletion of two A base pairs from a ten base pair poly A sequence at nucleotides 709 to 718 of the normal RII sequence, and addition of one or two A base pairs into the ten base pair poly A sequence at nucleotides 709-718 of the normal RII sequence. RII mutations arising from addition or deletion of one or two A bases within the poly A sequence at nucleotides 709-718 encode truncated RII related proteins that contain the RII ligand binding domain but lack the RII membrane anchoring domain so that they will be secreted from the cell into the blood.
Mutant RII may be detected by assays performed on samples from the patient which may be samples from tumor or normal tissue or samples of biological fluids, including serum, plasma, effusions, ascites, urine, stool, cerebrospinal fluid, semen, breast aspirates and fluids of ovarian origin. Suitable assays include immunoassay using antibodies reactive with mutant RII or by assays sensitive to DNA or RNA sequence. Truncated secreted mutant RII proteins may be detected by their ability to bind to TGFxcex2. Mutant RII proteins may be detected by TGFxcex2 binding to soluble proteins or abnormally sized proteins or reaction of such a soluble protein with antibody specific for the external domain of wild type RII. Alternatively, the presence of mutant RII in a patient may be detected by immunoassays which detect, in the patient, production of an antibody response aimed against either the RII mutation or, due to breaking of immune tolerance, against other epitopes on the native RII protein.
In yet another embodiment, this invention provides a method to aid in diagnosing cancer in a patient comprising detecting a mutant RII receptor for TGFxcex2 in a sample of biological fluid from the patient. The mutant form of RII may be detected directly or it may be detected indirectly by detecting antibody immunologically reactive with the mutant form of RII in a sample of biological fluid from the patient. The antibody immunologically reactive with mutant RII receptor for TGFxcex2 may also be immunologically reactive with wild type RII receptor.
In another embodiment, this invention provides a heterologous polynucleotide, either DNA or RNA, comprising a nucleotide sequence encoding a mutant form of TGFxcex2 receptor RII, the mutation being addition or deletion of one or two adenosine base-pairs from a ten base-pair poly-adenosine sequence at nucleotides 709 to 718 of the normal RII sequence, or preferably, a two base-pair insertion of GT which occurs in a six base pair repeat sequence GTGTGT at nucleotides 1931-1936 of the normal RII sequence. In a particular embodiment, this heterologous polynucleotide is an expression vector which expresses an inactive mutant form of RII.
In still another embodiment, this invention provides a therapeutic method for treating a cancer patient, wherein neoplastic cells of the patient express a non-functional mutant form of a growth regulatory gene, wherein the growth regulatory gene encodes a type II receptor which is a member of a family of serine/threonine receptors which bind members of a family of TGFxcex2-like factors or the wild-type of the growth regulatory gene contains repetitive DNA sequence motifs in the coding region, the method comprising administering to the patient an immunogenic amount of an immunogenic composition comprising a non-functional mutant form of the growth regulatory gene product or an expression vector encoding the same non-functional mutant form of the growth regulatory gene as the neoplastic cells of the patient. In a particular embodiment, this invention provides a therapeutic method for treating a colon or gastric cancer patient whose tumor expresses a mutant form of RII by administering to the patient an immunogenic amount of an expression vector encoding mutant TGFxcex2 receptor RII which is the same mutant form of RH as expressed by the patient""s tumor cells.
In still another embodiment, this invention provides a mutant protein of human TGFxcex2 receptor RII substantially free of other human proteins, the mutant RII protein having a sequence encoded by RII cDNA altered by a mutation, the mutation being either deletion or insertion of one or two A base pairs in a ten base-pair poly A sequence at nucleotides 709 to 718 of the normal RII sequence, or preferably, a two base pair insertion of GT which occurs in a six base pair repeat sequence GTGTGT at nucleotides 1931-1936 of the normal RII sequence. This invention also provides antibodies specific for the mutant protein.
In yet another embodiment, this invention provides a therapeutic method for treating a colon cancer patient, where the patient""s tumor cells express a mutant form of RII, the mutation being a two base pair insertion of GT which occurs in a six base pair repeat sequence GTGTGT at nucleotides 1931-1936 of the normal RII sequence, or deletion or insertion of one or two A base-pairs in a ten base pair poly A sequence at nucleotides 709 to 718 of the normal RII sequence, comprising administering to the patient an immunogenic composition containing a mutant form of RII containing the same RII mutation as the tumor cells of the patient.
In yet another embodiment, this invention provides a therapeutic method for treating a cancer patient, wherein neoplastic cells of the patient express a non-functional mutant form of a growth regulatory gene, wherein the growth regulatory gene encodes a type II receptor which is a member of a family of serine/threonine receptors which bind members of a family of TGFxcex2-like factors or the wild-type of the growth regulatory gene contains repetitive DNA sequence motifs in the coding region, the method comprising administering to the patient an effective amount of an substance specifically immunoreactive with the non-functional mutant form of the growth regulatory gene expressed by the neoplastic cells of the patient, the substance being preferably antibodies specifically immunoreactive with the growth regulatory gene or specific activated cytotoxic immune cells. In a preferred embodiment, the therapeutic method comprises administering a substance specifically immunoreactive with RII, the type II receptor for TGFxcex2.
In still another embodiment, this invention provides a therapeutic method for treating a cancer patient, wherein neoplastic cells of the patient express a non-functional mutant form of a growth regulatory gene, wherein the growth regulatory gene encodes a type II receptor which is a member of a family of serine/threonine receptors which bind members of a family of TGFxcex2-like factors or the wild-type of the growth regulatory gene contains repetitive DNA sequence motifs in the wild-type coding region, the method comprising administering to the patient a gene therapy vector encoding a functional form of the growth regulatory gene expressed by the neoplastic cells of the patient, the gene being operably linked to a promoter, wherein said gene therapy vector is expressed in the patient to produce a functional form of the growth regulatory gene expressed by the neoplastic cells of the patient. In a particular embodiment, this invention provides a therapeutic method for treating a patient having colon cancer of the RER phenotype comprising administering to the patient a gene therapy vector encoding functional TGFxcex2 receptor RII operably linked to a promoter, wherein the gene therapy vector is expressed in the patient to produce functional RII.
In investigations to determine whether inactivation of TGFxcex2 receptors is a mechanism by which human colon cancer cells lose responsiveness to TGFxcex2, it was discovered that RII receptors were inactivated in a subset of colon cancer cell lines. Surprisingly, RII inactivation was a common characteristic of tumor cells exhibiting microsatellite instability (hereafter referred to as RER+, for xe2x80x9creplication errorsxe2x80x9d). Inactivation of RII, by frameshift mutations in regions of small repeat sequences, appears to be a critical step in tumor progression, rather than a simple correlate of the RER+ phenotype.